The characteristics and functions of cells are determined and maintained by cellular organelles and the cellular cytoskeleton. Cellular organelles include, but are not limited to, nucleus, mitochondria, peroxisomes, Golgi apparatus, lysosomes, endoplasmic reticulum, centrosome, and vacuoles. The term cytoskeleton (cytoskeletal structures) refers to an extensive scaffolding of fibrillar elements, including the three filamentous systems: microfilaments (or actin filaments), microtubules, and intermediate filaments. It may also include linin filaments. The components of the cytoskeleton are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. Among these organelles and cytoskeletal structures, centrosome, microtubules, mitochondrion, endoplasmic reticulum lysosomes, and nuclear envelope are most important.
The centrosome, a central body (or the major microtubule-organizing center (MTOC) of the cell) plays a key role in the temporal and spatial distribution of the interphasic and mitotic microtubule network. Therefore, the centrosome could be considered a major determinant of the overall organization of the cytoplasm and of the fidelity of cell division (Hsu, L. C. and White, R. L. (1998) Proc Natl Acad Sci USA 27;95(22):12983-8). Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division, once and only once in each cell cycle, are all ensured through the actions of tightly regulated centrosomal function (Tanaka, T., et al., (1999) Cancer Res 59(9): 2041-4). Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis, and determines the number, polarity, and organization of interphase and mitotic microtubules (Tanaka, T., et al., (1999) Cancer Res 59(9): 2041≧4; Pihan, G. A., et al., (1998) Cancer Res 58(17):3974-85). The main function of the centrosome is the nucleation of microtubules, and the controlled cycle of its duplication, the two duplicated entities functioning as mitotic spindle poles during subsequent cell division. Centrosomes and their associated microtubules direct events during mitosis and control the organization of animal cell structures and movement during interphase. Although the precise mechanisms by which duplicated chromosomes are equally segregated during mitosis are largely unknown, the centrosome is believed to play an important role(s) in the formation of bipolar spindles (Tanaka, T., et al., (1999) Cancer Res 58(17):3974-85). The microtubule nucleating ability of centrosomes of tissue sections is retained even after several years of storage as frozen tissue blocks (Salisbury, J. L., et al., (1999) J. Histochem. Cytochem. 47(10):1265-74).
In animal cells, the centrosome is composed of two centrioles surrounded by the so-called pericentriolar material (PCM), which consists of a complex thin filament network and two sets of appendages.
Malignant tumors generally display abnormal centrosome profiles, characterized by an increase in size and number of centrosomes, by their irregular distribution, abnormal structure, aberrant protein phosphorylation, and by increased microtubule nucleating capacity in comparison to centrosomes of normal tissues (Lingle, W. L. et al., (1998) Proc Natl Acad Sci USA 95(6): 2950-5; Xu, X., et al., (1999) Mol Cell 3(3):389-95; Brinkley, B. R., et al., (1998) Cell Motil Cytoskeleton 41(4):281-8; Doxsey, S. (1998) Nat Genet 20(2):104-6; Kuo, K. K., et al., (2000) Hepatology 31(1):59-64). Among the abnormalities, centrosome hyperamplification is found to be more frequent in a variety of tumor types (Carroll, P. E., et al., (1999) Oncogene 18;18(11):1935-44; Hinchcliffe, E. H., et al., (1999) Science 283(5403):851-4; Xu, X., et al., (1999) Mol Cell 3(3):389-95).
Centrosome consists of many key proteins such as, SKP1p, cyclin-dependent kinase 2-cyclin E (Cdk2-E) (Hinchcliffe, E. H., et al., (1999) Science 283(5403): 851-4), kendrin (Flory, M. R., et al., (2000) Proc Natl Acad Sci USA 23;97(11):5919-23), Protein kinase C-theta (Passalacqua, M., et al., (1999) Biochem J 337(Pt 1): 113-8), EB1 protein. Recently, a variety of cell cycle-regulated kinases or tumor suppressor genes are located in or are core components of the centrosome. They include Nek2 (Fry, A. M., et al., (1999) J Biol Chem 274(23): 1304-10), protein kinase A type II isozymes (Keryer, G., et al., (1999) Exp Cell Res 249(1):131-146), heat shock Cognate 70 (HSC70) (Bakkenist, C. J., et al., (1999) Cancer Res 59(17):4219-21), PH33 (Nakadai, T., et al., (1999) J Cell Sci 112 (Pt9):1353-64), AIKs (Kimura, M., et al., (1999) J Biol Chem 274(11)7334-40), human SCF(SKP2) subunit p19(SKP1) (Gstaiger, M., et al., (1999) Exp Cell Res 247(2)554-62), STK15/BTAK (Zhou, H., et al., (1998) Nat Genet 20(2): 189-93), C-Nap1 (Fry, A. M., et al., (1998) J Cell Biol 274(23): 1304-10), Tau-like proteins (Cross, D., et al., (1996) Exp Cell Res 229(2):378-87), cyclin E (Carroll, P. E., et al., (1999; Mussman, J. G., et al., (2000) Oncogene 23;19(13):1635-46), p53, retinoblastoma protein pRB and BRCA1(Hsu, L. C., et al., (1998) Proc Natl Acad Sci USA 95(22):12983-8). These proteins are required in the initiation of DNA replication and mitotic progression (Gstaiger, M., et al., (1999) Exp Cell Res 15;247(2):554-62).
Microtubules, a filamentous system, are linear polymers of alpha- and beta (the beta1, beta2, and beta4 isotypes)-tubulin heterodimers. Except for being a frame of cellular membrane and organelles, microtubules may play an important role in other aspects. Microtubules are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. Microtubules are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis (Shan, B., et al., (1999) Proc Natl Acad Sci USA 96(10):5686-5691). They are nucleated by centrosome through the kinetochores of the centrosome. The spindle is a microtublule-based superstructure that assembles during mitosis to separate replicated DNA. Chromosome attachment to and movement on the spindle is intimately tied to the dynamics of microtubule polymerization and depolymerization. The sister chromatid pairs must maintain a stable attachment to spindle microtubules as the microtubules interconvert between growing and shrinking states. Drugs that are currently used in cancer therapy were designed to perturb microtubule shortening (depolymerization) or lengthening (polymerization) (Compton, D. A., et al., (1999) Science 286:913-914).
Other cytoskeletons such as membrane skeleton, microvilli, cilia, flagella, microfilaments, actin filaments, contractile ring, and intermediate filaments are all important in the organization of the cytoplasm and of the fidelity of cell division.
In addition to the centrosome and microtubules, other cellular organelles or cellular sub organelles such as mitochondrion, chromosomes, chromatin, nuclei, nuclear matrix, nuclear lamina, core filaments, nuclear envelope (NEs), nuclear pore complexes (NPCs), nuclear membrane, centrioles, pericentriolar material (PCM), mitotic spindle, spindle pole bodies (SPBs), contractile rings, proteasomes, telomere, plasma membranes, Golgi complexes, Golgi apparatus, endoplasmic reticulum (ER), endosomes, peroxisomes, proteasomes, phagosomes, ribosomes, are all important in maintaining a cell's life. Endoplasmic reticulum, e.g. is the site of synthesis and maturation of proteins.
Therefore, identification of a novel less-costing, simple, and effective method for the visualization of cellular organelles and/or cytoskeleton is indeed necessary in cell biology, cell cycle, signal transduction, development biology, and cancer research.
However, most available methods for the visualization of the centrosome and other important cellular organelles and/or cytoskeleton are based on the antigen-antibody reaction (Lingle, W. L. et al., (1998) Proc Natl Acad Sci USA 95(6): 2950-5; Xu, X., et al., (1999) Mol Cell 3(3):389-95; Brinkley, B. R., et al., (1998) Cell Motil Cytoskeleton 41(4):281-8; Doxsey, S. (1998) Nat Genet 20(2):104-6; Carroll, P. E., et al., (1999) Oncogene 18;18(11):1935-44; Hinchcliffe, E. H., et al., (1999) Science 283(5403):851-4; Xu, X., et al., (1999) Mol Cell 3(3):389-95). These techniques have been proved to be very costly, poorly reproducible, time consuming, and requiring of very strict conditions. Particularly, these methods can not be used to demonstrate the dynamic states of cells.
It is against this background, this invention provides a biochemical method for visualizing cellular organelles and/or cytoskeletons, by treating tissues or cells with crystallizing agents. The crystallizing agents or compounds used in this invention are a variety of tetrazolium salts. The cell-mediated reduction of some tetrazolium salts has long been used as a cell number-counting method (Berridge, M V, and Tan, A S., (1993) Arch Biochem Biophys 303(2): 474-482; Bernas, T., et al., (1999) Biochim Biophys Acta 12;1451(1):73-81; Abe, K., and Saito, H., (1999) Brain Res 29;830(1):146-54; Liu, Y., et al., (1997). J Neurochem 69(2):581-93; Abe, K., and Saito, H., (1998) Neuroscience Res 31:29-305). In the visualization of cellular organelles and/or cytoskeletons, the application of tetrazolium salts has never been mentioned. The inventor of this invention has found that tetrazolium salts can specifically concentrate on cellular organelles and/or cytoskeletons of a variety of cells and tissues, with the formation of visible crystals in these places. The visualization of cellular organelles and/or cytoskeletons using a biochemical approach instead of the complicated immune methods provides a less costly, very simple, quick, and effective method for the visualization of cellular organelles and/or cytoskeleton. It provides a tool with great potential in studying cell biology, structural biology, cell cycle, signal transduction, development biology, and oncology.